Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps

<div><p>To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins <i>Mp</i>AFP and <i>Mh</i>Lap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 μm/s, the <i>Mp</i>AFP extender domain fails at an unfolding force <i>F</i>_u = 348 ± 37 pN and <i>Mh</i>Lap at <i>F</i>_u = 306 ± 51 pN in buffer with 10 mM Ca²⁺. These forces place both extender domains well above the mechanical stability of many other β-sandwich domains in mechanostable proteins. We propose that the increased stability of <i>Mp</i>AFP and <i>Mh</i>Lap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.</p></div

Schematic representation of full adhesins <i>Mp</i>AFP and <i>Mh</i>Lap and corresponding SMFS constructs.

<p>The mechanical stability of region II of the full adhesin <i>Mp</i>AFP (1.5 MDa) and <i>Mh</i>Lap (0.3 MDa) (A) is investigated using octameric constructs (B). Region II of <i>Mp</i>AFP consists of 120 identical 104-amino-acid repeats, whereas region II of <i>Mh</i>Lap consists of 25 repeats of 97 amino acids, with on average 76% sequence identity between subsequent repeats. <i>Mp</i>AFP RII₈-GFP consists of eight <i>Mp</i>AFP RII repeats separated into two sections of tetra-tandemers by a GFP protein included in the middle which serves as internal force calibration standard. <i>Mh</i>Lap RII₈ consists of repeats 2–5 and 21–24. Both constructs have two C-terminal cysteines to promote the interaction of the proteins with the gold surface. The full amino acid sequences of the protein constructs are given in Section A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174682#pone.0174682.s001" target="_blank">S1 Supporting Information</a>. The pickup of the adhesin construct <i>Mp</i>AFP RII₈-GFP by the AFM tip is shown schematically in (C).</p

Pulling speed dependence of <i>Mp</i>AFP RII and unfolding force histograms of <i>Mp</i>AFP RII, <i>Mh</i>Lap RII and I27.

<p>(A) A linear dependence of <i>Mp</i>AFP RII unfolding force with pulling speed is visible. The measured unfolding forces for I27 at 1 μm/s pulling speed were in agreement with data of I27 taken from Brockwell <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174682#pone.0174682.ref032" target="_blank">32</a>], Carrion-Vazquez <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174682#pone.0174682.ref005" target="_blank">5</a>] and Fowler <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174682#pone.0174682.ref033" target="_blank">33</a>]. (B) Normalized histograms of measured unfolding forces of I27 (N = 349), <i>Mh</i>Lap RII (N = 1006) and <i>Mp</i>AFP RII (N = 518) at 1 μm/s pulling speed.</p

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps

<div><p>To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins <i>Mp</i>AFP and <i>Mh</i>Lap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 μm/s, the <i>Mp</i>AFP extender domain fails at an unfolding force <i>F</i>_u = 348 ± 37 pN and <i>Mh</i>Lap at <i>F</i>_u = 306 ± 51 pN in buffer with 10 mM Ca²⁺. These forces place both extender domains well above the mechanical stability of many other β-sandwich domains in mechanostable proteins. We propose that the increased stability of <i>Mp</i>AFP and <i>Mh</i>Lap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.</p></div

<i>Mp</i>AFP RII unfolding force depends on Ca²⁺ concentration.

<p>(A) Overlay of force curves of <i>Mp</i>AFP RII₈-GFP at 10 mM and 30 μM Ca²⁺. Force peaks of <i>Mp</i>AFP RII are on average ~100 pN lower when the protein is in 30 μM calcium compared to the force peaks of <i>Mp</i>AFP RII in 10 mM calcium. Force peak of GFP is unaffected, which is as expected since no Ca²⁺ ions are bound to GFP. (B) Overlay of four force curves of <i>Mp</i>AFP RII₈-GFP in 10 mM Ca²⁺ (left) and 30 μM Ca²⁺ (right). A larger spread in unfolding force peaks is observed for <i>Mp</i>AFP RII at 30 μM Ca²⁺ compared to <i>Mp</i>AFP RII in 10 mM Ca²⁺. All force measurements were performed at 1 μm/s pulling speed.</p

Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities

Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications

Typical sawtooth-like force-extension curves.

<p>Force curves of (A) <i>Mp</i>AFP RII₈-GFP, (B) <i>Mh</i>Lap RII₈ and (C) I27^RS₈ were obtained at a pulling speed of 1 μm/s and 10 mM Ca²⁺. The worm-like chain (WLC) model was applied to analyze the observed unfolding peaks from which we obtain values for a contour length increase Δ<i>L</i>_c upon unfolding and a persistence length <i>L</i>_p. The force-distance curve of the <i>Mp</i>AFP RII₈-GFP displays seven peaks corresponding to the unfolding of RII monomers (red WLC fit) and a much smaller peak at a small extension corresponding to GFP unfolding (green WLC fit). At a 1 μm/s pulling speed we obtained Δ<i>L</i>_c = 33.2 ± 3 nm, Δ<i>L</i>_c = 33.6 ± 5 nm and Δ<i>L</i>_c = 27.3 ± 5 nm for <i>Mp</i>AFP RII, <i>Mh</i>Lap RII and I27, respectively. Experimental data are shown in black; red and green solid lines correspond to WLC fits.</p